CN116637068A - Preparation method and application of biohydrogel for promoting bone defect repair - Google Patents

Abstract

The application discloses a preparation method and application of biological hydrogel for promoting bone defect repair, comprising the following steps of dissolving SDF-1and BMP-2 in PBS; dissolving 4-arm-PEG-NH2 and ODEX in a mixed solution of SDF-1and BMP-2 respectively; mixing 4-arm-PEG-NH2 solution and ODEX solution, and rotating; standing to obtain the biological hydrogel. According to the technical scheme, the biological hydrogel carrying the cytokines is used for being placed into the bone defect, so that the repairing and treating effects of the bone defect are improved; the preparation method is mainly prepared by crosslinking 4-arm amino polyethylene glycol and oxidized dextran through Schiff base reaction, and SDF-1and BMP-2 are prepared in the material preparation process. SDF-1 can induce bone marrow mesenchymal stem cells to homing, so that bone marrow mesenchymal stem cells directionally migrate to bone defect sites, BMP-2 is a classical cytokine which strongly promotes proliferation and differentiation of bone marrow mesenchymal stem cells into bone, and PEG-ODEX hydrogel is an excellent drug carrier and provides a good stable environment for positioning and releasing SDF-1and BMP-2. The biological hydrogel has the effect of repairing bone defect.

Description

A preparation method and application of biological hydrogel for promoting bone defect repair

technical field

The invention relates to the field of medicine, in particular to a method for preparing a biological hydrogel for promoting bone defect repair.

Background technique

As the world's population is aging more and more serious, musculoskeletal diseases such as fractures and osteoporosis are increasing rapidly,

Consequently, medical costs associated with musculoskeletal disorders continue to increase. In the United States, there are more than 620,000 cases of bone grafts required for large bone defects caused by cancer and trauma each year, costing about $2.5 billion. At present, the main surgical treatment is the filling of metal implants. However, due to metal implant wear and improper loading, the prosthesis loosens and shifts, inflammation around the prosthesis occurs, bone resorption and osteolysis, etc., and the final outcome is graft failure. Autologous bone grafting can be used to treat large bone defects. It is considered the gold standard for the treatment of large bone defects. However, bone grafting still has shortcomings. For autologous bone grafting, the bone tissue must be taken out from the healthy donor site of the patient, and then implanted into the diseased site, which will prolong the operation time; secondly, the autologous bone graft is mainly taken from bone tissue such as the pelvis or ilium, which may occur in a certain period of time. To a certain extent, it causes more serious complications at the donor site, and only a small amount of bone tissue can be obtained from the donor site. Bone allografts refer to bone tissue obtained from other individuals in humans, such as human cadavers and donors. Bone allografts may present risks of immune rejection, reduced biological activity, and pathogen transmission. The natural insufficiency of surgical metal implants and auto/allografts has prompted an in-depth exploration of bone tissue engineering alternatives.

The most important biological elements required for bone tissue engineering include seed cells, extracellular matrix scaffolds, and cytokines that promote growth, differentiation, and angiogenesis. The focus of bone tissue engineering is to develop implants with appropriate porosity to support and provide space for cells to crawl and adhere, thereby promoting bone tissue repair and regeneration. An ideal bone tissue engineering implant should have a porous structure with a three-dimensional network, so the three-dimensional porous scaffold can be used as a temporary scaffold to guide the growth and regeneration of new tissue. For optimal bone regeneration, implants should degrade over time and be replaced by host bone tissue, which remodels according to load and function. The rate of degradation should be similar to the rate of bone growth. Implants should meet certain mechanical conditions and deliver cytokines and biological signals in a specific way. The implant can also serve as a storehouse or carrier for various growth factors, cytokines and cell transplantation. Although local injection of cytokine therapy has shown good results in clinical applications, the factors diffuse outside the injury site and have low repair potential. Polymer scaffolds can address this issue by providing a confined space for cytokines to localize at the injury site and release them sustainably.

To sum up, how to prepare a new type of implant material to improve the effect of repairing and treating bone defects is an urgent problem to be solved by those skilled in the art.

Contents of the invention

The main purpose of the embodiments of the present invention is to propose a biohydrogel preparation method to promote bone defect repair, aiming to design a new type of implant material preparation method for improving the effect of bone defect repair and treatment.

The technical solution of the present invention to solve the above-mentioned technical problems is to provide a method for preparing a biological hydrogel that promotes bone defect repair, comprising the following steps:

Dissolve SDF-1 and BMP-2 in PBS;

Dissolve 4-arm-PEG-NH2 and ODEX in the mixed solution of SDF-1 and BMP-2 respectively;

Mix the 4-arm-PEG-NH2 solution and the ODEX solution and swirl;

Stand still to obtain biological hydrogel.

In an embodiment of the present invention, in the step of dissolving SDF-1 and BMP-2 in PBS, the solubility of SDF-1 and BMP-2 is 50 μg/L-2000 μg/L.

In one embodiment of the present invention, in the step of mixing and rotating the 4-arm-PEG-NH2 solution and the ODEX solution, the weight ratio of the 4-arm-PEG-NH2 solution to the ODEX solution is 2:1 .

In one embodiment of the present invention, the steps of dissolving 4-arm-PEG-NH2 and ODEX in the mixed solution of SDF-1 and BMP-2 respectively include:

Take 4-arm-PEG-OH and dissolve it in dichloromethane;

Add methanesulfonyl chloride and triethylamine to the 4-arm-PEG-OH solution, and continue to stir the reaction;

Precipitate the solution of methanesulfonyl chloride and triethylamine with ice anhydrous ether, and dry it in vacuum at 45°C to obtain a dry powder;

The dry powder was reacted with ammonia water for 7 days, and after cooling down to room temperature, the aqueous phase was extracted with dichloromethane, concentrated under reduced pressure, and dropped into cold ether to obtain 4-arm-PEG-NH2.

In one embodiment of the present invention, the step of dissolving 4-arm-PEG-NH2 and ODEX in the mixed solution of SDF-1 and BMP-2 respectively also includes:

Dissolve dextran in distilled water;

Sodium periodate was added to the dextran solution, and the stirring reaction was continued for 24 hours;

Dialyze the reacted solution with deionized water;

The dialyzed solution was freeze-dried to obtain ODEX.

In order to solve the above-mentioned technical problems, the present invention also proposes the application of the bio-hydrogel prepared according to the above-mentioned bio-hydrogel preparation method for promoting bone defect repair in bone defect repair.

According to the technical scheme of the present invention, the application uses biological hydrogel loaded with cytokines to insert bone defects and improve the effect of repairing and treating bone defects; it is mainly composed of 4-arm aminopolyethylene glycol (4-arm poly (ethyleneglycol)amine,4-arm-PEG-NH2) and oxidized dextran (ODEX) were cross-linked by Schiff base reaction, and loaded with stromal cell-derived factor- 1 (StromalCell Derived Factor-1, SDF-1) and bone morphogenetic protein-2 (Bone Morphogenetic Proteins-2, BMP-2). In this application, SDF-1 can induce bone marrow mesenchymal stem cells (bone marrow stromal cells, BMSCs) homing, and make bone marrow mesenchymal stem cells migrate to the bone defect site through directional chemotaxis. Cytokines for the proliferation and differentiation of mesenchymal stem cells into bone, PEG-ODEX hydrogel provides a good and stable carrier environment for the release of SDF-1 and BMP-2. It has been verified by experiments that the biohydrogel has the effect of repairing bone defects, and can be used as a new scaffold for bone tissue based on bone tissue cells.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained according to the structures shown in these drawings without creative effort.

Fig. 1 is the schematic diagram of the biological hydrogel prepared by the biological hydrogel preparation method for bone defect repair according to the present invention;

Fig. 2 is a schematic diagram of gelation of the biohydrogel preparation method for repairing bone defects of the present invention;

Fig. 3 is the schematic diagram of electron microscope scanning result of biological hydrogel of the present invention;

Fig. 4 is the schematic diagram of the rheological experiment result of biohydrogel of the present invention;

Figure 5 is a schematic diagram of the results of different concentrations of SDF-1 of the present invention on BMSCs chemotaxis;

Fig. 6 is a schematic diagram of the effect of different concentrations of BMP-2 of the present invention on the proliferation of BMSCs;

Figure 7 is a schematic diagram of the staining results of living and dead cells after the biological hydrogel of the present invention is co-cultured with BMSCs;

Figure 8 is a schematic diagram of the detection results of CCK-8 after co-cultivation of biological hydrogel and BMSCs of the present invention:

Figure 9 is a graph of the in vivo degradation rate of the empty hydrogel of the present invention;

Figure 10 is a schematic diagram of the in vitro drug release kinetics of the biohydrogel of the present invention;

Figure 11 is a schematic diagram of semi-quantitative analysis of alizarin red staining of biological hydrogels of the present invention;

Figure 12 is a schematic diagram of micro-CT results and analysis of the biohydrogel of the present invention to promote bone defect repair

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of the present invention.

It should be noted that all directional indications (such as up, down, left, right, front, back...) in the embodiments of the present invention are only used to explain the relationship between the components in a certain posture (as shown in the accompanying drawings). Relative positional relationship, movement conditions, etc., if the specific posture changes, the directional indication will also change accordingly.

In addition, in the present invention, descriptions such as "first", "second" and so on are used for description purposes only, and should not be understood as indicating or implying their relative importance or implicitly indicating the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "several" and "plurality" mean at least two, such as two, three, etc., unless otherwise specifically defined.

In the present invention, unless otherwise specified and limited, the terms "connection" and "fixation" should be understood in a broad sense, for example, "fixation" can be a fixed connection, a detachable connection, or an integral body; It can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediary, and it can be an internal communication between two elements or an interaction relationship between two elements, unless otherwise clearly defined. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention according to specific situations.

In addition, the technical solutions of the various embodiments of the present invention can be combined with each other, but it must be based on the realization of those skilled in the art. When the combination of technical solutions is contradictory or cannot be realized, it should be considered as a combination of technical solutions. Does not exist, nor is it within the scope of protection required by the present invention.

The present invention proposes a preparation method of biological hydrogel for promoting bone defect repair, and aims to design a novel composite implant material for improving the effect of bone defect repair and treatment.

A plurality of embodiments are set as follows, wherein in all embodiments, experimental reagents and equipment are as follows:

Experimental reagents: 4-arm-PEG-OH, dichloromethane, methanesulfonyl chloride, triethylamine, anhydrous ether, ammonia water, dextran, sodium periodate were purchased from Aladdin, DMEM/F12 complete medium, fetal bovine Serum (FBS), rabbit bone marrow mesenchymal stem cells, PBS, SDF-1 and BMP-2 freeze-dried powder were all purchased from Gibco, CCK8 kit, live dead staining kit, and Alizarin red staining kit were purchased from Beijing Bi Yuntian, SDF-1 and BMP-2 Elisa kits were purchased from Jiangyuan Biotech.

Experimental equipment: OHAUS-Adventure electronic balance is from Ohaus Company in the United States; MilliporeDirect-Q8 UV ultrapure water machine is from Merck Millipore, Germany; MS7-H550-S constant temperature heating magnetic stirrer and dialysis bag are from Obtained from Beijing Dalong Xingchuang Experimental Instrument Co., Ltd.; the ultra-low temperature refrigerator is obtained from Thermo Scientific; the LABCONCO freeze dryer is obtained from the US LABCONCO company.

The specific structure of the biohydrogel preparation method for promoting bone defect repair proposed by the present invention will be described in specific examples below:

Example 1

A method for preparing a biological hydrogel for promoting bone defect repair, comprising the following steps:

Step 1. Preparation of 4-arm-PEG-NH2

(1) Weigh 20.0g of 4-arm-PEG-OH and dissolve it in 100mL of dichloromethane.

(2) Then add 2.3g of methanesulfonyl chloride and 1.1g of triethylamine to the solution in (1), and continue to stir and react for 24h;

(3) After the reaction is completed, the solution in (2) is precipitated with ice anhydrous ether, and then vacuum-dried at 45° C. until the weight is constant;

(4) React the dry powder obtained in (3) with 100mL ammonia water for 7 days, and after cooling down to room temperature, extract the water phase with dichloromethane, concentrate it to 50mL under reduced pressure, and drop it into cold ether to prepare 4-arm -PEG-NH2, stored in -20°C refrigerator.

Step 2. Preparation of ODEX

(1) Weigh 2.0g dextran into a 100mL dry flask, add distilled water at room temperature and stir slowly until the dextran dissolves;

(2) Add 792 mg of sodium periodate to the solution in (1), and continue stirring for 24 hours at room temperature;

(3) Dialyze the solution of (2) with deionized water for 3 days (molecular weight 3500Da) (for removing crude product);

(4) The solution in (3) was frozen overnight at -80°C, then transferred to a freeze dryer for 24 hours, and finally the solution was freeze-dried to obtain ODEX, which was stored at -20°C.

Step 3, preparation of SDF-1 and BMP-2 mixed solution

(1) Dissolve SDF-1 and BMP-2 in PBS.

Understandably, the concentration of SDF-1 and BMP-2 is 50 μg/L-2000 μg/L

The concentrations of SDF-1 and BMP-2 used in this step are: 0 μg/L, 20 μg/L, 50 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 1000 μg/L, 2000 μg/L, prepared into Cell culture media with different concentrations of SDF-1 are used for Transwell experiments on BMSCs to verify the optimal concentration of SDF-1 for chemotaxis of BMSCs, and are formulated into cell culture media with different concentrations of BMP-2 to cultivate BMSCs. The CCK-8 method was used to analyze the proliferation of BMSCs, and the results are shown in Figure 5. When the concentration of SDF-1 was 500 μg/L, the chemotactic effect on BMSCs was the most obvious; the results were shown in Figure 6. When the concentration of BMP-2 was 500μg/L, the proliferation effect on BMSCs was the most obvious.

Preferably, the solubility of both SDF-1 and BMP-2 is 500 μg/L.

4. Preparation of biohydrogels

(1) The final product prepared in step 1 and step 2 is dissolved in the mixed solution of SDF-1 and BMP-2 in step 3 respectively with 10wt%;

(2) Mix the 4-arm-PEG-NH2 solution of (1) with the ODEX solution at a weight ratio of 2:1. After rotating for 10s, add 400 μL of the mixture into a 24-well plate and place it for 15 minutes to obtain a biohydrogel ,As shown in Figure 1.

Understandably, the weight ratio of the 4-arm-PEG-NH2 solution to the ODEX solution is 2:1. Since the reaction of 4-arm-PEG-NH2 and ODEX is rapid, therefore, it is necessary to dissolve 4-arm-PEG-NH2 and ODEX in the mixed solution of SDF-1 and BMP-2 respectively, in the 4-arm - When PEG-NH2 and ODEX react, the formed hydrogel porous fibers can directly physically wrap SDF-1 and BMP-2, and then form a biohydrogel, as shown in Figure 2.

In this application, the biological hydrogel loaded with cytokines is used to insert bone defects to improve the repair and treatment effect of bone defects; it is mainly prepared by cross-linking 4-arm-PEG-NH2 and ODEX through Schiff base reaction , and loaded with SDF-1 and BMP-2 during material preparation. SDF-1 can induce the homing of BMSCs and make bone marrow mesenchymal stem cells migrate to the bone defect site by chemotaxis. BMP-2 is a classic cytokine that strongly promotes the proliferation and differentiation of bone marrow mesenchymal stem cells into bone. PEG-ODEX water The gel provides a good and stable carrier environment for the release of SDF-1 and BMP-2. It has been verified by experiments that the hydrogel has the effect of repairing bone defects, and can be used as a new scaffold for bone tissue based on bone tissue cells.

Example 2

A method for preparing a biological hydrogel for promoting bone defect repair, comprising the following steps:

Step 1. Preparation of 4-arm-PEG-NH2:

(1) Weigh 20.0g of 4-arm-PEG-OH and dissolve it in 100mL of dichloromethane.

(2) Then add 2.3g of methanesulfonyl chloride and 1.1g of triethylamine to the solution in (1), and continue to stir and react for 24h;

(3) After the reaction is completed, the solution in (2) is precipitated with ice anhydrous ether, and then vacuum-dried at 45° C. until the weight is constant;

(4) React the dry powder obtained in (3) with 100mL ammonia water for 7 days, and after cooling down to room temperature, extract the water phase with dichloromethane, concentrate it to 50mL under reduced pressure, and drop it into cold ether to prepare 4-arm -PEG-NH2, stored in -20°C refrigerator.

Step 2. Preparation of ODEX:

(1) Weigh 2.0g dextran into a 100mL dry flask, add distilled water at room temperature and stir slowly until the dextran dissolves;

(2) Add 792 mg of sodium periodate to the solution in (1), and continue stirring for 24 hours at room temperature;

(3) Dialyze the solution of (2) with deionized water for 3 days (molecular weight 3500Da) (for removing crude product);

(4) The solution in (3) was frozen overnight at -80°C, then transferred to a freeze dryer for 24 hours, and finally the solution was freeze-dried to obtain ODEX, which was stored at -20°C.

Step 3. Preparation of no-load hydrogel:

(1) Dissolving the final product prepared in step 1 and step 2 in PBS with 10 wt% respectively;

(2) Mix the 4-arm-PEG-NH2 solution of (1) with the ODEX solution at a weight ratio of 2:1. After rotating for 10 seconds, add 400 μL of the mixture into a 24-well plate and place it for 15 minutes to obtain no-load hydrogel glue.

Step 4. Preparation of biological hydrogel

(1) Dissolve SDF-1 and BMP-2 in PBS.

(2) Dissolving the final product prepared in step 1 and step 2 in the mixed solution of SDF-1 and BMP-2 in (1) with 10 wt% respectively;

(3) Mix the 4-arm-PEG-NH2 solution of (2) with the ODEX solution at a weight ratio of 2:1. After rotating for 10s, add 400 μL of the mixture into a 24-well plate and place it for 15 minutes to obtain a biohydrogel ,As shown in Figure 1.

Characterization of Biohydrogels:

(1) After the biological hydrogel prepared by the above method was frozen at -80° C. for 12 hours, the frozen sample was freeze-dried for 24 hours. Scanning electron microscopy and rheological experiments on lyophilized biohydrogels. As shown in Figure 3 and Figure 4, the hydrogel microscopically presents a cross-linked network porous structure with a pore size between 50 μm and 100 μm. This pore size is also the optimal pore size for bone tissue engineering bioimplants, and this dense porous structure ensures the loading and controlled release characteristics of small molecule drugs and protein drugs. The storage modulus G' of the hydrogel is greater than the corresponding loss modulus G", exhibiting the properties of a gel.

Biocompatibility of empty hydrogels with biological hydrogels and analysis of proliferation of BMSCs:

(1) BMSCs were cultured in a DMEM/F12 medium containing 10% fetal bovine serum at 37°C in a cell culture incubator containing 5% CO ₂ . BMSCs cultured to the third passage were used for the evaluation of all in vitro cell experiments.

(2) The experimental process was to inoculate the BMSCs cell suspension at a density of 1× ¹⁰⁴ /well in blank wells (control, Ctrl group), containing empty hydrogel (pure hydrogel, H group) and biological hydrogel Glue (pure hydrogelincorporated SDF-1and BMP-2, H/SDF-1/BMP-2 group) culture wells.

(3) After incubating in the incubator for 1 day and 3 days, prepare the working solution of calcein-AM/PI staining agent according to the instructions of the live dead cell staining kit for soaking the samples. After immersing each group of samples for 15 minutes at 4°C in the dark, the working solution was removed, washed twice with PBS to remove excess dye, and the cell viability was observed under a fluorescence microscope. As shown in Figure 7, the number of stained BMSCs living cells in each group showed a trend of increasing significantly from day 1 to day 3. At each time point, there was no significant difference in the number of dead cells between the three groups. It shows that both the empty hydrogel and biohydrogel have good biocompatibility.

(4) Cell counting kit (Cell counting kit-8, CCK-8) was used to detect the specific effect of the compound system on the proliferation of BMSCs. 1× ¹⁰⁴ BMSCs were inoculated into H group and H/SDF-1/BMP-2 group respectively.

(5) After 1, 3 and 7 days of co-cultivation with each group of hydrogels, experiments were performed at each time point. Replace the complete medium in each group of culture wells, add 10% of the medium volume of CCK-8 working solution into the culture wells, and incubate at 37°C and 5% CO2 for 2 hours. The absorbance of each reaction solution was detected at 450 nm by a microplate reader. The results are shown in Figure 8, the BMSCs cells in each group showed a significant cell proliferation trend within the experimental period of 7 days, and the cell activity was good. Among them, on the 3rd and 7th days, there was no difference in the proliferation rate between the blank hole group and the empty hydrogel group, and the cell proliferation in the biological hydrogel group was more obvious, far exceeding the rest of the groups; The proliferation activity was not affected, while the biohydrogel loaded with SDF-1 and BMP-2 could promote the proliferation of BMSCs. It not only proves that the empty hydrogel has good biocompatibility, but also proves that the biohydrogel loaded with SDF-1 and BMP-2 can promote the proliferation of BMSCs.

(6) In the third step of the embodiment, the concentrations of the mixed solutions of SDF-1 and BMP-2 are: 0 μg/L, 20 μg/L, 50 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 1000 μg /L, 2000μg/L, prepared into cell culture medium containing different concentrations of SDF-1 and BMP-2 for subsequent experiments. The main effect of SDF-1 on BMSCs is chemotaxis, so Transwell experiments are used here to explore the optimal concentration of SDF-1. BMSCs were cultured in serum-free medium for 24 hours to starve them. 1000 starved BMSCs were planted in Transwell chambers, and each chamber was placed in medium containing different concentrations of SDF-1. The bottom of the small chamber is a layer of polycarbonate membrane, which is permeable and separates the culture fluid of the small chamber from the culture fluid of the orifice plate, and the culture fluid in the orifice plate can affect the cells in the small chamber. After 12 hours, cells will pass through the pores to the back of the polycarbonate membrane under the chemotaxis of SDF-1. Take out the small chamber, wipe off the residual cells on the front, fix the cells on the back with formaldehyde, stain with 2% gentian violet for half an hour, and observe the number of cells under a microscope. The results are shown in Figure 5, when the concentration is 500 μg/L, 1000 μg/L, 2000 μg/L, the chemotaxis effect is obvious. The effect of BMP-2 on the proliferation of BMSCs was detected by CCK-8 method, and the results are shown in Figure 6. When the concentration of BMP-2 was 500 μg/L and 1000 μg/L, the proliferation of BMSCs was most obvious. Considering cost and safety, preferably, the concentrations of SDF-1 and BMP-2 are respectively 500 μg/L.

Example 3

The application of biohydrogels in bone defect repair, including:

According to the steps of Example 2, no-load hydrogel and biological hydrogel were prepared;

In vivo degradation analysis of empty hydrogel:

(1) After SD rats were anesthetized with isoflurane, the hair on both sides of the back was shaved, and the hydrogel was injected subcutaneously, 0.4 mL on each side;

(2) Rats were sacrificed on days 0, 3, 7, 14, 21, 28, and 35. The skin around the injection was removed, the fascia was removed, and the residual hydrogel was weighed to observe the degradation. The results are shown in Figure 9. The hydrogel swelled after absorbing water in the early stage, and then degraded smoothly. The whole process lasted for about 35 days, achieving a long-term degradation and meeting the needs of osteogenesis. Therefore, when the biohydrogel is administered in the body, it can be degraded and meets the medication standard.

Drug Release Analysis of Biohydrogels:

(1) The drug release of biohydrogels in vitro was detected by Elisa method. The drug concentration loaded into the gel was increased to 100μg/L when testing the drug release in vitro. After the biohydrogel was prepared, it was placed in deionized water at 37°C, and the soaking solution was collected at 1, 2, 3, 5, 7, 14, 21, and 28 days, respectively. After collecting, add an equal amount of deionized water to continue soaking.

(2) Prepare the working solution according to the instructions of the Elisa kit, mix the soaking solution collected in (1) with the working solution, add it to the well plate with the antibody on the bottom of the kit, incubate for 30 minutes, and develop the color by After the reaction, the absorbance of each sample was measured at a wavelength of 450nm, and the drug concentration in the soaking solution was calculated according to the standard curve to evaluate the release of the drug. The results are shown in Figure 10, the drug release kinetics of H/SDF-1 and BMP-2 biohydrogels during 35 days, which shows a relatively good sustained drug release. The release rate of the drug was faster at the initial stage, and the total release percentages of SDF-1 and BMP-2 were 12.8%±1.2% and 15.7%±1.6% in the first day, respectively. In the next 28 days, the release rate of the drug Ming slowed down, showing a good sustained-release effect. However, by day 28 and day 35, the total amount of drug released from the biohydrogel showed little increase, so the collection of drug extract was stopped. In the sustained drug release architecture, the drug release process is mainly mediated by the degradation of the material and the diffusion of the drug itself. In the initial stage of release, since the hydrogel has not yet degraded, the drug is mainly released in the form of free diffusion. At the same time, the rate of drug diffusion is closely related to the pore size of the hydrogel. The larger the pore size, the easier it is for the drug to diffuse through the network structure of the hydrogel, thereby accelerating drug release. The pore size of the hydrogel is about 50-100 μm. Although the pore size is small, its ability to absorb water and swell is relatively strong, making it easier for SDF-1 and BMP-2 to diffuse to the outside. However, the burst release stage is not long. As the hydrogel swells to the limit and the hydrogel gradually degrades, the release of the drug becomes mainly dominated by the slow degradation of the material, so the release rate of the drug slows down, showing a good performance. Drug release profile. Overall, the biological hydrogel system constructed in this application can achieve good drug release results.

Effects of empty hydrogel and biohydrogel on osteogenic differentiation of BMSCs:

(1) The effect of biohydrogel on the osteogenic differentiation of BMSCs was evaluated and detected under the induction of osteogenic induction medium. BMSCs were seeded in Ctrl, H and H/SDF-1/BMP-2 well plates at a density of 1×10 ⁵ cells/well, and cultured with osteogenic induction medium.

(2) Alizarin red staining was performed on the 21st day after induction, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde at 37°C for 30 minutes. Alizarin red solution was then added to the fixed cells and incubated at room temperature for 30 minutes.

(3) Dissolve the calcified nodules in each group with 10% cetylpyridinium chloride solution, and detect the absorbance of the reaction solution at 562 nm for semi-quantitative analysis. The results are shown in Figure 9, at day 21, the H/SDF-1/BMP-2 group showed the deepest staining of calcium nodules and the largest staining area among all groups. The semi-quantitative analysis of alizarin red staining further confirmed the above experimental results, indicating that the number of calcified nodules in the H/SDF-1/BMP-2 group was significantly higher than that in the other groups. Osteoblast differentiation is a continuous process, and differentiated cells can secrete a mineralized extracellular matrix matrix, thereby promoting the deposition of minerals (such as calcium). Thus, calcium deposition is a marker of mature osteoblasts. Alizarin red staining results showed that the biohydrogel promoted the osteogenic differentiation of BMSCs in vitro.

Bone defect repair analysis of empty hydrogel and biohydrogel:

(1) Thirty New Zealand white rabbits were randomly divided into 3 groups, 10 in each group. Experimental animals were anesthetized with 3% (w/v) pentobarbital at a dose of 50 mg/kg before surgery. The skin was prepared after anesthesia, the operative area was disinfected, a drape was spread, and a longitudinal incision was made on the radial side of the distal left forelimb to separate the fascia and muscle layer by layer to expose the radius.

(2) Use a small electric saw to resect the 2cm long radius, resulting in a 2cm defect, and clean up the 0.5cm periosteum above and below the defect. In the Ctrl group, the wound was sutured layer by layer directly, in the H group, the wound was sutured layer by layer after injection of blank hydrogel at the defect site, and in the H/SDF-1/BMP-2 group, the wound was sutured layer by layer after injection of biological hydrogel at the defect site. Each experimental animal received intramuscular injection of penicillin (1.5 mg/kg) within 3 days after operation to prevent infection.

(3) At 12 weeks after the operation, the rabbits were euthanized under anesthesia, and the radius specimens were collected for micro-CT detection. The results are shown in Figure 12, the defects in the H/SDF-1 and BMP-2 groups were almost filled by new bone, and the BV/TV reached 90.2%±1.3%, while the new bone filling in the Ctrl group and H group was poor. Only a small amount of new bone was seen in the defect, and the broken end was in the shape of a mouse tail, forming atrophic nonunion. Therefore, biohydrogels can well promote bone defect repair, and biohydrogels can be applied to bone defect repair.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present invention can easily think of changes or Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (6)

1. A method for preparing a biological hydrogel for promoting bone defect repair, which is characterized by comprising the following steps:

dissolving SDF-1and BMP-2 in PBS;

dissolving 4-arm-PEG-NH2 and ODEX in a mixed solution of SDF-1and BMP-2 respectively;

mixing 4-arm-PEG-NH2 solution and ODEX solution, and rotating;

standing to obtain the biological hydrogel.

2. The method of claim 1, wherein the step of dissolving SDF-1and BMP-2 in PBS, wherein the solubility of SDF-1 is 50-2000 μg/L and the concentration of BMP-2 is 50-2000 μg/L.

3. The method of claim 2, wherein in the step of mixing and spinning the 4-arm-PEG-NH2 solution and the ODEX solution, the weight ratio of the 4-arm-PEG-NH2 solution to the ODEX solution is 2:1.

4. The method of claim 1, wherein the step of dissolving 4-arm-PEG-NH2 and ODEX in the mixed solution of SDF-1and BMP-2, respectively, comprises:

dissolving 4-arm-PEG-OH in dichloromethane;

adding methylsulfonyl chloride and triethylamine into the 4-arm-PEG-OH solution, and continuing stirring for reaction;

precipitating the solution of methylsulfonyl chloride and triethylamine with ice anhydrous diethyl ether, and vacuum drying at 45 ℃ to obtain dry powder;

and (3) reacting the dry powder with ammonia water for 7 days, cooling to room temperature, extracting the water phase with dichloromethane, concentrating under reduced pressure, and dripping into cold diethyl ether to obtain 4-arm-PEG-NH2.

5. The method of claim 1, wherein the step of dissolving 4-arm-PEG-NH2 and ODEX in the mixed solution of SDF-1and BMP-2, respectively, further comprises:

dissolving dextran in distilled water;

adding sodium periodate into the dextran solution, and continuing stirring for reaction for 24 hours;

dialyzing the reacted solution by deionized water;

freeze-drying the dialyzed solution to obtain ODEX.

6. Use of the bio-hydrogel prepared by the method for preparing a bio-hydrogel for bone defect repair according to any one of claims 1 to 5 in bone defect repair.